Patients with end stage organ failure require organ transplantation for survival. The major limiting factor in clinical transplantation is the shortage of suitable human donors. Over the past ten years the size of the waiting list of patients for organs has increased dramatically, from approximately 30,000 in 1991 to approximately 80,000 in 2001 (Source: New York Organ Donor Network; Association of Organ Procurement Organizations' Death Record Review Study from 1997 to 1999, provided by 30 organ procurement organizations). Despite this increasing need over the past ten years, the number of organ donations has remained flat (approximately 20,000 per year).
According to the United Network for Organ Sharing (UNOS) as of Jul. 17, 2003, there were 82,249 patients waiting for organ transplants in the United States. The need for specific organs was as follows:
Kidney55,133Liver17,304Pancreas1,413Kidney and2,378PancreasIntestine173Heart3,717Heart-Lung184Lung3,912
Across the U.S., an average of 17 men, women and children of all races and ethnic backgrounds die every day for lack of donated organs, thus, each year, more than 6,200 Americans die waiting for an organ transplant. A need for a more reliable and unlimited source of organs has led to investigation of the potential for transplantation of organs from other animals, referred to as xenotransplantation.
Pigs are considered the most likely source of xenograft organs. The supply of pigs is plentiful, breeding programs are well established, and their size and physiology are compatible with humans. Xenotransplantation, however, presents its own set of problems. The most significant is immune rejection. The first immunological hurdle is “hyperacute rejection” (HAR). HAR can be defined by the ubiquitous presence of high titers of pre-formed natural antibodies binding to the foreign tissue. The binding of these natural antibodies to target epitopes on the donor organ endothelium is believed to be the initiating event in HAR. This binding, within minutes of perfusion of the donor organ with the recipient blood, is followed by complement activation, platelet and fibrin deposition, and ultimately by interstitial edema and hemorrhage in the donor organ, all of which cause failure of the organ in the recipient (Strahan et al. (1996) Frontiers in Bioscience 1, e34-41).
Except for Old World monkeys, apes and humans, most mammals carry glycoproteins on their cell surfaces that contain galactose alpha 1,3-galactose (Galili et al., J. Biol. Chem. 263: 17755-17762, 1988). Humans, apes and Old World monkeys have a naturally occurring anti-alpha gal antibody that is produced in high quantity (Cooper et al., Lancet 342:682-683, 1993). It binds specifically to glycoproteins and glycolipids bearing galactose alpha-1,3 galactose.
In contrast, glycoproteins that contain galactose alpha 1,3-galactose are found in large amounts on cells of other mammals, such as pigs. This differential distribution of the “alpha-1,3 GT epitope” and anti-Gal antibodies (i.e., antibodies binding to glycoproteins and glycolipids bearing galactose alpha-1,3 galactose) in mammals is the result of an evolutionary process which selected for species with inactivated (i.e. mutated) alpha-1,3-galactosyltransferase in ancestral Old World primates and humans. Thus, humans are “natural knockouts” of alpha1,3GT. A direct outcome of this event is the rejection of xenografts, such as the rejection of pig organs transplanted into humans initially via HAR.
A variety of strategies have been implemented to eliminate or modulate the anti-Gal humoral response caused by porcine xenotransplantation, including enzymatic removal of the epitope with alpha-galactosidases (Stone et al., Transplantation 63: 640-645, 1997), specific anti-gal antibody removal (Ye et al., Transplantation 58: 330-337,1994), capping of the epitope with other carbohydrate moieties, which failed to eliminate alpha-1,3-GT expression (Tanemura et al., J. Biol. Chem. 27321: 16421-16425, 1998 and Koike et al., Xenotransplantation 4: 147-153, 1997) and the introduction of complement inhibitory proteins (Dalmasso et al., Clin.Exp.Immunol. 86: 31-35, 1991, Dalmasso et al. Transplantation 52:530-533 (1991)). C. Costa et al. (FASEB J 13, 1762 (1999)) reported that competitive inhibition of alpha-1,3-GT in H-transferase transgenic pigs results in only partial reduction in epitope numbers. Similarly, S. Miyagawa et al. (J. Biol. Chem 276, 39310 (2001)) reported that attempts to block expression of gal epitopes in N-acetylglucosaminyltransferase III transgenic pigs also resulted in only partial reduction of gal epitopes numbers and failed to significantly extend graft survival in primate recipients.
Single allele knockouts of the alpha-1,3-GT locus in porcine cells and live animals have been reported. Denning et al. (Nature Biotechnology 19: 559-562, 2001) reported the targeted gene deletion of one allele of the alpha-1,3-GT gene in sheep. Harrison et al. (Transgenics Research 11: 143-150, 2002) reported the production of heterozygous alpha-1,3-GT knock out somatic porcine fetal fibroblasts cells. In 2002, Lai et al. (Science 295: 1089-1092, 2002) and Dai et al. (Nature Biotechnology 20: 251-255, 2002) reported the production of pigs, in which one allele of the alpha-1,3-GT gene was successfully rendered inactive. Ramsoondar et al. (Biol of Reproduc 69, 437-445 (2003) reported the generation of heterozygous alpha-1,3-GT knockout pigs that also express human alpha-1,2-fucosyltransferase (HT), which expressed both the HT and alpha-1,3-GT epitopes.
PCT publication No. WO 94/21799 and U.S. Pat. No. 5,821,117 to the Austin Research Institute; PCT publication No. WO 95/20661 to Bresatec; and PCT publication No. WO 95/28412, U.S. Pat. No. 6,153,428, U.S. Pat. No. 6,413,769 and US publication No. 2003/0014770 to BioTransplant, Inc. and The General Hospital Corporation provide a discussion of the production of alpha-1,3-GT negative porcine cells based on knowledge of the cDNA of the alpha-1,3-GT gene (and without knowledge of the genomic organization or sequence). However, there was no evidence that such cells were actually produced prior to the filing date of these applications and the Examples were all prophetic.
The first public disclosure of the successful production of a heterozygous alpha-1,3-GT negative porcine cell occurred in July 1999 at the Lake Tahoe Transgenic Animal Conference (David Ayares, et al., PPL Therapeutics, Inc.). Prior to the present invention, no one had published or publicly disclosed the production of a homozygous alpha 1,3GT negative porcine cell. Further, since porcine embryonic stem cells have not been available to date, there was and still is no way to use an alpha-1,3-GT homogygous embryonic stem cell to attempt to prepare a live homogygous alpha-1,3-GT knock out pig.
On Feb. 27, 2003, Sharma et al. (Transplantation 75:430-436 (2003) published a report demonstrating a successful production of fetal pig fibroblast cells homozygous for the knockout of the alpha-1,3-GT gene.
PCT publication No. WO 00/51424 to PPL Therapeutics describes the genetic modification of somatic cells for nuclear transfer. This patent application discloses the genetic disruption of the alpha-1,3-GT gene in porcine somatic cells, and the subsequent use of the nucleus of these cells lacking at least one copy of the alpha-1,3-GT gene for nuclear transfer.
U.S. Pat. No. 6,331,658 to Cooper & Koren claims but does not confirm any actual production of genetically engineered mammals that express a sialyltransferase or a fucosyltransferase protein. The patent asserts that the genetically engineered mammals would exhibit a reduction of galactosylated protein epitopes on the cell surface of the mammal.
PCT publication No. WO 03/055302 to The Curators of the University of Missouri confirms the production of heterozygous alpha 1,3GT knockout miniature swine for use in xenotransplantation. This application is generally directed to a knockout swine that includes a disrupted alpha-1,3-GT gene, wherein expression of functional alpha-1,3-GT in the knockout swine is decreased as compared to the wildtype. This application does not provide any guidance as to what extent the alpha-1,3-GT must be decreased such that the swine is useful for xenotransplantation. Further, this application does not provide any proof that the heterozygous pigs that were produced exhibited a decreased expression of functional alpha1,3GT. Further, while the application refers to homozygous alpha 1,3GT knockout swine, there is no evidence in the application that any were actually produced or producible, much less whether the resultant offspring would be viable or phenotypically useful for xenotransplantation.
Total depletion of the glycoproteins that contain galactose alpha 1,3-galactose is clearly the best approach for the production of porcine animals for xenotransplantation. It is theoretically possible that double knockouts, or the disruption of both copies of the alpha 1,3GT gene, could be produced by two methods: 1) breeding of two single allele knockout animals to produce progeny, in which case, one would predict based on Mendelian genetics that one in four should be double knockouts or 2) genetic modification of the second allele in a cell with a pre-existing single knockout. In fact, this has been quite difficult as illustrated by the fact that while the first patent application on knock-out porcine cells was filed in 1993, the first homozygous alpha 1,3GT knock out pig was not produced until July 2002 (which was based on the work of the present inventor and described herein).
Transgenic mice (not pigs) have historically been the preferred model to study the effects of genetic modifications on mammalian physiology, for a number of reasons, not the least of which is that mouse embryonic stem cells have been available while porcine embryonic stem cells have not been available. Mice are ideal animals for basic research applications because they are relatively easy to handle, they reproduce rapidly, and they can be genetically manipulated at the molecular level. Scientists use the mouse models to study the molecular pathologies of a variety of genetically based diseases, from colon cancer to mental retardation. Thousands of genetically modified mice have been created to date. A “Mouse Knockout and Mutation Database” has been created by BioMedNet to provide a comprehensive database of phenotypic and genotypic information on mouse knockouts and classical mutations Brandon et al Current Biology 5[7]:758-765 (1995); Brandon et al Current Biology 5[8]:873-881 (1995), this database provides information on over 3,000 unique genes, which have been targeted in the mouse genome to date.
Based on this extensive experience with mice, it has been learned that transgenic technology has some significant limitations. Because of developmental defects, many genetically modified mice, especially null mice created by gene knock out technology die as embryos before the researcher has a chance to use the model for experimentation. Even if the mice survive, they can develop significantly altered phenotypes, which can render them severely disabled, deformed or debilitated (Pray, Leslie, The Scientist 16 [13]: 34 (2002); Smith, The Scientist 14[15]:32, (2000); Brandon et al Current Biology 5[6]:625-634 (1995); Brandon et al Current Biology 5[7]:758-765 (1995); Brandon et al Current Biology 5[8]:873-881 (1995). Further, it has been learned that it is not possible to predict whether or not a given gene plays a critical role in the development of the organism, and, thus, whether elimination of the gene will result in a lethal or altered phenotype, until the knockout has been successfully created and viable offspring are produced.
Mice have been genetically modified to eliminate functional alpha-1,3-GT expression. Double-knockout alpha-1,3-GT mice have been produced. They are developmentally viable and have normal organs (Thall et al. J Biol Chem 270:21437-40(1995); Tearle et al. Transplantation 61:13-19 (1996), see also U.S. Pat. No. 5,849,991). However, two phenotypic abnormalities in these mice were apparent. First, all mice develop dense cortical cataracts. Second, the elimination of both alleles of the alpha-1,3-GT gene significantly affected the development of the mice. The mating of mice heterozygous for the alpha-1,3-GT gene produced genotype ratios that deviated significantly from the predicted Mendelian 1:2:1 ratio (Tearle et al. Transplantation 61:13-19 (1996)).
Pigs have a level of cell surface glycoproteins containing galactose alpha 1,3-galactose that is 100-1000 fold higher than found in mice. (Sharma et al. Transplantation 75:430-436 (2003); Galili et al. Transplantation 69:187-190 (2000)). Thus, alpha1,3-GT activity is more critical and more abundant in the pig than the mouse.
Despite predictions and prophetic statements, prior to this invention, no one knew whether the disruption of both alleles of the alpha-1,3-GT gene would be lethal or would effect porcine development or result in an altered phenotype (Ayares et al. Graft 4(1)80-85 (2001); Sharma et al. Transplantation 75:430-436 (2003); Porter & Dallman Transplantation 64:1227-1235 (1997); Galili, U. Biochimie 83:557-563 (2001)). Indeed, many experts in the field expressed serious doubts as to whether homozygous alpha-1,3-GT knockout pigs would be viable at all, much less develop normally. Such concerns were expressed up until the double knockout pig of the present invention was produced. Examples of statements by those working in the field at the time included the following.
“The abundantly expressed alpha-gal epitope may have some biological roles in pig development, such as in cell-cell interaction. If this assumption is correct, pigs may not develop in the absence of this epitope (Galili, U. Biochimie 83:557-563 (2001).”
“The inability to generate knockout pigs for alpha-gal may suggest that alpha-gal epitopes are indispensable in this species (Galili et al. Transplantation 69:187-190 (2000)).”
“Although double-knockout alpha-gal mice develop and remain fairly normal, the possibility exists that deletion of this enzyme could have more severe consequences in other animals (Porter & Dallman Transplantation 64:1227-1235 (1997)).”
“It is possible that the GT(−/−) pig may not be viable because the GT gene is essential for embryonic development. An answer to this question and to the relevance of GT(−/−) pigs to xenotransplantation research must await, if possible, the production of the appropriate pigs (Sharma et al. Transplantation 75:430-436 (2003)).”
“Since Gal epitope expression in pig organs is up to 500-fold higher than in mouse organs, there is the possibility that alphaGT activity is more crucial to the pig and could effect development of these pigs (Ayares et al. Graft 4(1)80-85 (2001)).”
Thus, until a viable double alpha-1,3-GT knockout pig is produced, according to those of skill in the art at the time, it was not possible to determine (i) whether the offspring would be viable or (ii) whether the offspring would display a phenotype that allows the use of the organs for transplantation into humans.
It is therefore an object of the present invention to provide viable pigs which lack any expression of functional alpha1,3GT.
It is another object of the present invention to provide procine cells, tissues and organs, which lack any expression of functional alpha1,3GT, for use in xenotransplantation or other biomedical applications.
It is a further object of the present invention to provide a method to select and screen for porcine cells, which lack galactose alpha 1,3-galactose epitopes on the cell surface.